The present invention relates to novel nucleotides, and more specifically, ribonucleotide oligonucleotides (RNOs) that are specifically designed to inhibit viral replication at the intracellular sites of infection in the respiratory tract. The RNOs are capable of binding to both the negative and positive strands of influenza RNA segments, thereby inhibiting the virus"" ability to produce various viral components, and therefore inhibit viral propagation. The RNOs provided may be formulated in liposomes, which facilitate their therapeutic delivery to intracellular sites of infection, and additionally increase antiviral efficacies.
Despite recent advances in antiviral therapy and vaccination; acute respiratory infections caused by influenza viruses are important causes of human mortality and morbidity worldwide. Although influenza viruses generally cause a mild malaise in healthy individuals, for the high-risk segments of the population, such as the elderly, the young, and individuals with underlying chronic illness, influenza and the complications resulting from secondary infections, can be life threatening. It is estimated that in the United States alone, more than 10,000 people die each year from influenza and complications from influenza.
Vaccination, or immunoprophylaxis, using inactivated or killed whole virus is a widely used conventional preventative measure used to reduce the impact of influenza [Centers for Disease Control, Prevention and control of influenza: recommendations of the Immunization Practices Advisory Committee Weekly Report, 1991; 41 (RR-9) 1-5]. However, there are significant limitations and drawbacks in such an antiviral vaccination.
A major disadvantage with killed whole virus, or inactivated, influenza virus vaccinations is that they are ineffective against new virus variants that result from genetic changes, such as those changes that arise from antigenic shifts in the haemagglutinin (HA) protein resulting in variations from one HA subtype to another. In addition, because such virus vaccines are propagated in eggs, individuals or populations that are sensitive to eggs and egg products are unsuitable for such vaccination.
Another conventional preventative measure used to reduce or control the impact of influenza is chemoprophylaxis, or chemotherapy, with an influenza-specific antiviral drug, such as amantadine or rimantadine [Centers for Disease Control. Prevention and control of influenza: recommendations of the Immunization Practices Advisory Committee Weekly Report, 1991; 41 (RR-9) 1-5]. In addition, neuraminidase inhibitors have also been recently commercialized for the treatment of influenza.
There are however, limitations to the use of such anti-influenza drugs. In particular, anti-influenza drugs such as amantadine and rimantadine are only partially effective against influenza A viruses, and are essentially ineffective against B strains of the influenza virus. Moreover, there is a rapidly increasing number of influenza A viruses that are developing resistance to amantadine and rimantadine, whereby such antiviral drugs are becoming rapidly ineffective against these more resistant strains of influenza A virus [Bleshe, R. B., et al, (1989), J. Infect. Dis., 159: 430-435].
Oligonucleotides are promising therapeutic agents that have been shown to be effective against cancer and infectious diseases. The prior art also includes the use of antisense deoxyribonucleotides to treat neoplastic and infectious diseases [Mizuta, T., et al, (1999) Nature Biotechnology, 17 (6), 583-587]. Oligonucleotides directed against a target gene or messenger RNA can result in the inhibition of transcription and/or translation of virus genetic material, thereby blocking the synthesis of key virus proteins required for virus propagation. However, such unmodified oligonucleotides are particularly susceptible to degradation by nucleases in the serum, thus limiting their therapeutic potential.
Liposomes are microscopic lipid vesicles and they have been used successfully as drug delivery systems to enhance the therapeutic effectiveness of a number of therapeutic agents against influenza infections, including antiviral antibodies [Wong, J. P., et al, (1994) Immunology, 81:280-284], interferon-gamma [Saravolac, E. G., et al, (1996) Antiviral Res., 29:199-207] and ribavirin [Kende, M., et al, (1985) Antimirob. Agents Chemotherapy., 27:903-907].
It is known to use antisense deoxyribonucleotide oligonucleotides (i.e. DNA molecules) to treat viral infections. In addition, the known antisense oligonucleotides are adversely restricted to gene targets.
The present invention seeks to overcome the obstacles encountered in the prior art and provides the use of novel ribonucleotides, and liposome encapsulated ribonucleotides for the treatment of viral infection, and in particular influenza virus infection. The use of liposomes in this invention allows not only for the oligonucleotides be protected by nuclease degradation, but also allows the oligonucleotides to be delivered to the intracellular sites of infection in the lungs, thereby resulting in enhancing their antiviral inhibition efficacy.
Thus, an object of the present invention is to provide ribonucleotide oligonucleotides (RNOs), either free, or encapsulated in liposomes for the prevention and treatment of viral infections. The use of RNOs offers several distinct advantages over antisense deoxyribonucleotides oligonucleotides. For example, influenza virus, which is an RNA virus, replicates itself by transcribing its RNA into mRNA and then proceeds to translate mRNA into viral proteins. Subsequent to such protein synthesis, the replication of the viral RNA takes place via a synthesis of the cRNA. Accordingly, the use of RNOs, provided according to the present invention, will allow for the binding of such RNOs to either the viral RNA, cRNA and/or mRNA, therefore allowing for the increased functional efficacy in the treatment and prevention of viral infection.
An object of the present invention is to provide a method for treating, preventing and/or controlling influenza virus infections.
A particular object of the present invention is to provide novel ribonucleotides (RNOs) sequences specifically designed to inhibit viral replication and proliferation at intracellular sites of infection.
Another object of the present invention is to provide novel RNOs specifically designed to inhibit influenza virus replication and proliferation at intracellular sites of infection.
The present invention provides a plurality of ribonucleotide oligonucleotides (RNOs) with specific sequences, which act individually or collectively as therapeutic agents in the treatment and prevention of viral infection, and more specifically, influenza viral infection.
Accordingly, one aspect of the present invention provides a ribonucleotide having the following sequence:
ribonucleotide JW-T1 represented by oligonucleotide sequence SEQ ID NO: 1;
ribonucleotide JW-T2 represented by oligonucleotide sequence SEQ ID NO: 2;
ribonucleotide JW-T3 represented by oligonucleotide sequence SEQ ID NO: 3;
Reference will also be made to the following negative control sequence:
ribonucleotide JW-T4 represented by oligonucleotide sequence SEQ ID NO: 4;
wherein RNO JW-T4 does not inhibit the virus, and is provided for use in control studies relating to the present invention.
In addition, the present invention provides a ribonucleotide sequence including any one of RNOs JW-T1, JW-T2, or JW-T3, or any equivalent sequence thereof, or any combination thereof. Therefore, the present invention is not limited by RNOs sequences JW-T1, JW-T2, or JW-T3 represented in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, but embodies any equivalent sequences, and larger fragment sequences, that may be used in accordance with the preferred uses of the present invention.
A preferred embodiment of the present invention comprises the formulation of such RNOs, either individually, or in any combination thereof, into liposomes.
Although the RNOs of the present invention are preferably directed to the treatment of influenza viral infection, the invention contemplates the use of ribonucleotides for the prevention and treatment of all viral infections. More specifically, the specific RNOs provided in the present invention are directed to a preferred use of preventing and treating all viral infections wherein the virus comprises haemagglutinin or a haemagglutinin gene. More preferably, the specific RNOs of the present invention may preferably be used for the prevention and treatment of influenza viral infection.
Therefore, all viral infections that allow for the binding of the RNOs, namely, JW-T1, JW-T2, or JW-T3, or comprise any equivalent sequence thereof, or any other sequence comprising or incorporating such RNOs, whereby the binding of such sequences to a virus result in the inhibition of viral infection, are embodied in the additional uses of the present invention.
Moreover, the present invention also embodies any RNO sequence equivalent to the preferred RNO sequences of the present invention, namely, JW-T1, JW-T2, or JW-T3. For example, the present invention also comprises RNO sequences that are effectively equivalent to the preferred RNOs sequences provided, such that mutations or changes effected to such preferred RNOs to yield modified, but equivalent RNO sequences are included in the embodiments of the present invention.
Appropriately, RNO, when referred to herein, includes RNO sequences JW-T1, JW-T2, or JW-T3, or any equivalent sequence thereof, or any larger sequence comprising JW-T1, JW-T2, or JW-T3, or any equivalent sequence thereof.
The present invention thus provides novel RNOs which are effective as either sense and/or antisense oligonucleotide sequences, capable of binding either the plus strand and/or the negative strand of a virus, in particular an influenza virus, thereby inhibiting the virus"" ability to replicate.
The genetic sequences of the novel RNOs provided are unique and distinct from previously described antisense oligonucleotides. Moreover, the present invention provides the use of ribonucleotides for the effective treatment of viral infections. The RNOs provided in the present invention may bind to either sense and/or antisense oligonucleotide sequences, binding either the plus strand and/or the negative strand of a virus. Therefore, the RNOs of the present invention may bind to viral RNA, viral mRNA, and/or viral cRNA, and therefore, allow for the exceptional inhibition of viral infection.
It is well known that haemagglutinin (HA) plays an important and essential role in the pathogenesis of influenza viral infection. Accordingly, in a preferred embodiment, the novel RNOs provided act as either sense and/or antisense oligonucleotides capable of binding to the haemagglutinin (HA) gene, thereby inhibiting HA gene transcription, and/or are capable of binding to the transcribed HA mRNA, thereby inhibiting HA protein synthesis, and/or are capable of binding to the HA protein of the influenza virus particle itself, thereby inhibiting the action of HA protein, and effectively inhibiting viral replication and proliferation.
In another embodiment of the present invention, the novel RNOs are formulated or encapsulated into liposomes, whereby the encapsulation of such therapeutic RNOs into liposomes facilitates the RNOs delivery to, and efficacy at, a site of infection.
Liposome encapsulation of RNOs provides additional benefits. For example, liposome encapsulation will facilitate delivery of the active compounds to the sites of infection. In addition, liposome encapsulation may be used to attenuate the potential toxicity of RNOs as well as potentiate their antiviral efficacy. Moreover, liposome encapsulation may also allow for a reduction in the dosage of such active compounds, thereby reducing any toxic or side effects that may develop due to high dosage concentrations.
According to preferred embodiments of the present invention, RNOs JW-T1, JW-T2, and JW-T3, or any oligonucleotide sequence comprising JW-T1, JW-T2, and JW-T3, or any equivalent sequence or compound thereof, may be encapsulated into liposomes, and preferably, into non-charged liposomes, such that their antiviral efficacies may be optimized. Anionic and cationic liposomes may be used for liposome encapsulation.
The present invention also comprises the use of individual antiviral RNOs, either free or liposome encapsulated, or combinations of antiviral RNOs, either free or liposome encapsulated, according to embodiments of the present invention, for the treatment of influenza viral infection, and more specifically, influenza A viral infection in mammals.
In addition, the present invention provides therapeutic formulations of such RNOs, either free or liposome encapsulated, wherein said formulations may be designed for aerosol delivery, or may be formulated into a pharmaceutical composition that may optimize or facilitate its use.